Abstract
In the growing field of systems biology, the knowledge of protein concentrations is highly required to truly understand metabolic and adaptational networks within the cells. Therefore we established a workflow relying on long chromatographic separation and mass spectrometric analysis by data independent, parallel fragmentation of all precursor ions at the same time (LC/MS(E)). By prevention of discrimination of co-eluting low and high abundant peptides a high average sequence coverage of 40% could be achieved, resulting in identification of almost half of the predicted cytosolic proteome of the Gram-positive model organism Bacillus subtilis (>1,050 proteins). Absolute quantification was achieved by correlation of average MS signal intensities of the three most intense peptides of a protein to the signal intensity of a spiked standard protein digest. Comparative analysis with heavily labeled peptides (AQUA approach) showed the use of only one standard digest is sufficient for global quantification. The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell. To prove this method for its biological relevance selected physiological aspects of B. subtilis cells grown under conditions requiring either amino acid synthesis or alternatively amino acid degradation were analyzed. This allowed both in particular the validation of the adjustment of protein levels by known regulatory events and in general a perspective of new insights into bacterial physiology. Within new findings the analysis of "protein costs" of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MS(E)) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities.
Highlights
The quantification results covered almost four orders of magnitude, ranging roughly from 10 to 150,000 copies per cell
Within new findings the analysis of “protein costs” of cellular processes is extremely important. Such a comprehensive and detailed characterization of cellular protein concentrations based on data independent, parallel fragmentation in liquid chromatography/mass spectrometry (LC/MSE) data has been performed for the first time and should pave the way for future comprehensive quantitative characterization of microorganisms as physiological entities
The determination of protein costs of different biological processes using genome-scale absolute protein quantification should represent a major breakthrough in understanding bacterial physiology and cellular design
Summary
Bacterial Growth and Sample Preparation—B. subtilis BSB 168 trpϩ strain [20] was grown in minimal medium with glucose as carbon source (condition S, 2 g/l (NH4)2SO4, 1.4 g/l K2HPO4, 0.6 g/l KH2PO4, 0.1 g/l Na3citrate, 0.2 g/l MgSO4, 0.001 g/l MnSO4, 5 g/l glucose, 0.001 g/l FeCl3): and in defined amino acid based medium (condition CH, 10 g/l Caseinhydrolysate, 3.9 g/l L-glutamic acid, 1.3 g/l L-alanine, 1.5 g/l L-asparagine, 1.4 g/l KH2PO4, 1.4 g/l NH4Cl, 0.1 g/l Na2SO4, 0.1 g/l NH4NO3, 0.1 g/l MgSO4, 0.02 g/l CaCl2, 0.075 g/l MnSO4, 0.001 g/l FeCl3) as described previously [21]. LC-SRM for the four additional proteins was carried out with a reverse phase nanoHPLC (easy-nLCII, Thermo Fisher) using a 80 min gradient. For additional filtering of database search results, all proteins identified in only one of the nine replicates of one sample (three technical replicates of each of the three biological replicates) were discarded leading to a FDR between 3 and 4% on protein level These FDR thresholds were applied for the global analysis of the data. For comparison of the absolute quantification results between the AQUA and Hi3 method, the results were standardized (molecule per cell level) and a Wilcoxon-Rank-Sum test was applied
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